Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes. Most damage-induced mutagenesis in Escherichia coli is dependent upon the UmuD'C protein complex, which comprises DNA polymerase V (pol V). Pol V has intrinsically weak catalytic activity, but is dramatically stimulated by interactions with ATP and RecA to form pol V Mut. In a collaborative study with Michael Cox at the University of Wisconsin and Myron Goodman at the University of Southern California, we have recently identified the protein-protein interface between UmuC and RecA that is critical for pol V activation. This was achieved by using mutant RecA proteins, a range of in vitro and in vivo approaches, and a photo-activated unnatural amino acid at RecA N113. In particular, we identified a RecA surface defined by residues 112-117 that either directly interacts with, or is in very close proximity to, amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein therefore represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. We have extended our studies on the regulation of pol V through a collaboration with Antoine van Oijen and Andrew Robinson at the University of Wollongong in Australia. In particular, we focused on the hitherto under appreciated special regulation imposed on pol V in E.coli. Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are much less common in bacteria. Due to its mutagenic potential, pol V activity has previously been shown to be controlled by means of an elaborate regulatory system at both the transcriptional and posttranslational levels. However, by using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we demonstrated that pol V is also subject to a novel form of spatial regulation. After an initial delay ( 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD to UmuD&#8242;. Classic SOS damage response mutants either block umuD(K97A) or constitutively stimulate recA(E38K) UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD&#8242;C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template. Our studies on the eukaryotic TLS polymerases focused on Saccharomyces cerevisiae polymerase eta (pol eta). Pol eta is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved steric gate residue which functions to prevent incorporation of NMPs during DNA synthesis. In our collaborative study with Susana Cerritelli and Robert Crouch (NICHD), we demonstrated that the Phe35 residue of S.cerevisiae pol eta functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol iota enzyme, wild-type pol eta does not readily incorporate NMPs in vitro. In contrast, a pol eta F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol eta F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1delta) and the TLS polymerase, pol zeta (rev3delta), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201delta strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1delta rev3delta rad30-F35A rnh201delta strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesized that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol eta can compete with the cells replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol eta in replicating common fragile sites (CFS) in human cells.